1. Field of the Invention
In one embodiment, the present invention is directed to a transfection device for eukaryotic cells. In another embodiment, the present invention is directed to kits and formulations for cell transfection which are useful with the transfection device. Methods of cell transfection using the transfection device are also described.
2. Description of the Related Art
Gene transfection methods can be used to introduce nucleic acids into cells and are useful in studying gene regulation and function. High throughput assays that can be used to screen large sets of DNAs to identify those encoding products with properties of interest are particularly useful. Gene transfection is the delivery and introduction of biologically functional nucleic acids into a cell, such as a eukaryotic cell, in such a way that the nucleic acid retains its function within the cell. Gene transfection is widely applied in studies related to gene regulation, gene function, molecular therapy, signal transduction, drug screening, and gene therapy studies. As the cloning and cataloging of genes from higher organisms continues, researchers seek to discover the function of the genes and to identify gene products with desired properties. This growing collection of gene sequences requires the development of systematic and high-throughput approaches to characterizing gene products and analyzing gene function, as well as other areas of research in cell and molecular biology.
Both viral and non-viral gene carriers have been used in gene delivery. Viral vectors have been shown to have higher transfection efficiency than non-viral carriers, but the safety of viral vectors hampers its applicability (Verma I. M and Somia N. Nature 389 (1997), pp. 239–242; Marhsall E. Science 286 (2000), pp. 2244–2245). Although non-viral transfection systems have not exhibited the efficiency of viral vectors, they have received significant attention, because of their theoretical safety when compared to viral vectors. In addition, viral vector preparation is a complicated and expensive process, which limits the application of viral vectors in vitro. The preparation of non-viral carriers is simpler and more cost effective in comparison to preparation of viral carriers, making synthetic gene carriers desirable as transfection reagents in in vitro studies.
Most non-viral vectors mimic important features of viral cell entry in order to overcome cellular barriers, which are meant to prevent infiltration by foreign genetic material. Non-viral gene vectors, based on a gene carrier backbone, can be classified as a) lipoplexes, b) polyplexes, and c) lipopolyplexes. Lipoplexes are assemblies of nucleic acids with a lipidic component, which is usually cationic. Gene transfer by lipoplexes is called lipofection. Polyplexes are complexes of nucleic acids with cationic polymer. Lipopolyplexes comprise both a lipid and a polymer component. Often such DNA complexes are further modified to contain a cell targeting or an intracellular targeting moiety and/or a membrane-destabilizing component, for example, a viral protein or peptide or a membrane-disruptive synthetic peptide. Recently, bacteria and phages have also been described as shuttles for the transfer of nucleic acids into cells.
Most non-viral transfection reagents are synthetic cationic molecules and have been reported to “coat” the nucleic acid by interaction of the cationic sites on the cation and anionic sites on the nucleic acid. The positively-charged DNA-cationic molecule complex interacts with the negatively charged cell membrane to facilitate the passage of the DNA through the cell membrane by non-specific endocytosis. (Schofield, Brit. Microencapsulated. Bull, 51(1):56–71 (1995)). In most conventional gene transfection protocols, the cells are seeded on cell culture devices 16 to 24 hours before transfection. The transfection reagent (such as a cationic polymer carrier) and DNA are usually prepared in separate tubes, and each respective solution is diluted in medium (containing no fetal bovine serum or antibiotics). The solutions are then mixed by carefully and slowing adding one solution to the other while continuously vortexing the mixture. The mixture is incubated at room temperature for 15–45 minutes to allow the transfection reagent-DNA complexes to form. Prior to transfection, the cell culture medium is removed and the cells are washed with buffer to remove the residues of serum and antibiotics. The solution containing DNA-transfection reagent complexes is added to the cells, and the cells are incubated for about 3–4 hours. The medium containing transfection reagent would then be replaced with fresh medium. The cells would finally be analyzed at one or more specific time point(s). This is obviously a time consuming procedure, particularly when the number of samples to be transfected is very large.
Several major problems exist in conventional transfection procedures. First, conventional procedures are time-consuming, particularly when there are many cell or gene samples to be used in transfection experiments. Also, the results derived from common transfection procedures are difficult to reproduce, due to the number of steps required. For instance, in producing the DNA-transfection reagent, the formation of the complex is influenced by concentration and volume of nucleic acid and reagents, pH, temperature, type of buffer(s) used, length and speed of vortexing, incubation time, and other factors. Although the same reagents and procedure may be followed, different results may be obtained. Results derived from multi-step procedures are often influenced by human or mechanical error or other variations at each step. In addition, refreshing the cell culture medium following transfection disturbs the cells and may cause them to detach from the surface on which they are cultured, thus leading to variation and unpredictability in the final results. Due to all the factors noted, conventional transfection methods require a highly skilled individual to perform the transfection experiment or assay.
Sabatini (U.S. 2002/0006664A1) describes DNA containing a mixture and deposited on a glass slide. However the system only allows transfection with the previously deposited DNA.
U.S. patent application Ser. No. 10/341,059, which is incorporated herein by reference, describes a cell culture/transfection device where the transfection is mediated by a lipid polymer. Some of the methods of application Ser. No. 10/341,059 may be applied to the transfection formulations described herein.
In one aspect of the present invention, a cell culture/transfection device is disclosed which reduces the cost of transfection assays. Transfection with a molecule of interest is accomplished in a simple procedure.
As discussed above, conventional transfection is a lengthy and technically difficult procedure. Generally, three steps are required: 1) cells are seeded in cell culture plate or dish and incubated until sufficient confluence is achieved; 2) transfection reagent/nucleic acid complexes are prepared; and 3) nucleic acids of interest are added along with the transfection reagent and further incubation is carried out. Two incubation periods are needed and typically it takes more than two days to complete all the steps. In contrast, embodiments of the present invention provide a simple procedure that involves only a single incubation step. A cell culture device, which has previously been coated with a transfection reagent, allows transfection by adding the nucleic acid of interest and the cell culture in succession. The transfected cells may then be cultured in the same device. Thus the cells may be transfected and cultured in the cell culture device without the need for further manipulation of the cells immediately after the transfection step. Transfection efficiency is comparable to regular transfection. Consequently, this invention provides an easy transfection procedure and reduces the time required for the transfection procedure by more than one day. Large numbers of cells may be transfected efficiently.